PROJECT SUMMARY During multicellular development, cells establish and maintain stable identities by activating lineage-specifying genes. Epigenetic mechanisms, involving the polycomb repressive system and its associated histone H3 lysine 27 tri-methylation (H3K27me3) modification, are critical for proper cell lineage specification, and are frequently disrupted in cancer; however, despite much work in many systems, it remains unclear how exactly these histone modifications control gene expression, and how their disruption drives malignancy. These questions remain unanswered, because we lack methods to follow epigenetic processes in living cells. We recently developed a reporter system to analyze epigenetic control in the activation of Bcl11b, an essential transcription factor for T-cell lineage commitment. To definitively test whether Bcl11b activation is controlled by cis-epigenetic mechanisms acting at single loci, we generated a mouse, where two Bcl11b loci are tagged with different fluorescent proteins (Ng et al. 2018). By following progenitors from these mice, we found that two Bcl11b alleles turn on independently in the same cell, with one allele often turning on multiple days before another. This work demonstrates that an epigenetic switch, acting independently on two Bcl11b loci, regulates the dynamics of gene activation and T-cell commitment. Here, in this proposal, we seek to elucidate the epigenetic mechanism controlling this lineage commitment switch, and determine impact of its disruption for leukemia initiation. We will test the hypothesis that repressive H3K27me3 modifications uphold a key control point for Bcl11b activation, and that disrupting this process can delay lineage commitment and drive leukemia. To do so, we will first define the role for H3K27me3 loss in controlling Bcl11b activation (Aim 1). To do so, we will perturb H3K27me3 modifications on the Bcl11b locus, and measure effects on locus activation dynamics. In these assays, the dual-color Bcl11b reporter strain provides a powerful tool to visualize control by epigenetic mechanisms in living cells. Next, we will determine how transcription factors work initiate H3K27me3 loss and gene activation (Aim 2). To do so, we will perturb candidate TFs and cis-regulatory regions on the Bcl11b locus, and determine resultant effects on H3K27me3 states and gene expression. Finally, we will determine whether delays in differentiation, caused by disruptions of epigenetic mechanisms, drive leukemia initiation (Aim 3). To do so, we will determine whether delayed Bcl11b activation slows down the pace of T-cell development, and whether this developmental slowdown can accelerate the onset of T-ALL in a mouse model. As polycomb mechanisms operate in diverse mammalian developmental processes, and because disruption of these mechanisms may be a major driver of malignancy, our findings could broadly impact diverse fields.